Peptides tend to be easily denatured due to their low stability, degraded by in-vivo proteolytic enzymes, thus losing the activity, and have a relatively small size, thereby easily passing through the kidney. Accordingly, in order to maintain the blood levels and the titers of a medicament comprising a peptide as a pharmaceutically effective component, it is necessary to administer the peptide drug frequently to a patient to maintain desired blood levels and titers. However, the peptide drugs are usually administered in the form of injectable preparations, and such frequent administration cause severe pain for the patients. To solve these problems, many efforts have been made. As one of such efforts, there has been an attempt to transfer the peptide drug through oropharyngeal or nasopharyngeal inhalation by increasing the transmission of the peptide drug through the biological membranes. However, this approach is still difficult in maintaining the in-vivo activity of the peptide drug due to the low in-vivo transfer efficiency, as compared to the injection.
On the other hand, many efforts have been made to improve the blood stability of the peptide drug, and to maintain the drug in the blood at a high level for a prolonged period of time, thereby maximizing the pharmaceutical efficacy of the drug. The long acting preparation of such peptide drug therefore needs to increase the stability of the peptide drug, and to maintain the titers at sufficiently high levels without causing immune responses in patients.
As a method for stabilizing the peptide, and inhibiting the degradation by a proteolytic enzyme, some trials have been performed to modify a specific amino acid sequence which is sensitive to the proteolytic enzyme. For example, GLP-1 (7-37 or 7-36 amide), which functions to reduce the glucose concentration in blood for treating a Type 2 diabetes, has a short half-life of the physiological activity of about 4 minutes or less (Kreymann et al., 1987), due to loss of the titers of GLP-1 through the cleavage between the 8th amino acid (Ala) and the 9th amino acid (Asp) by a dipeptidyl peptidase IV (DPP IV). As a result, various investigations have been made on a GLP-1 analog having resistance to DPP IV, and trials have been made for substitution of Ala8 with Gly (Deacon et al., 1998; Burcelin et al., 1999), or with Leu or D-Ala (Xiao et al., 2001), thereby increasing the resistance to DPP IV, while maintaining the activity. The N-terminal amino acid, His7 of GLP-1 is critical for the GLP-1 activity, and serves as a target of DPP IV. Accordingly, U.S. Pat. No. 5,545,618 describes that the N-terminus is modified with an alkyl or acyl group, and Gallwitz, et al. describes that 7th His was subject to N-methylation, or alpha-methylation, or the entire His is substituted with imidazole to increase the resistance to DPP IV, and to maintain physiological activity.
In addition to these modifications, an exendin-4, which is a GLP-1 analog purified from the salivary gland of a glia monster (U.S. Pat. No. 5,424,686), has resistance to DPP IV, and higher physiological activity than GLP-1. As a result, it had an in-vivo half-life of 2 to 4 hours, which was longer than that of GLP-1. However, with the method for increasing the resistance to DPP IV only, the physiological activity is not sufficiently sustained, and for example, in the case of a commercially available exendin-4 (exenatide), it needs to be injected to a patient twice a day, which is still difficult for patients.
These insulinotropic peptides have a problem, usually in that the size of the peptide is small. Thus, they cannot be recovered in the kidney, and are then extracorporeally discharged. Accordingly, a method for chemically adding a polymeric substance having high solubility, such as polyethylene glycol (PEG), onto the surface of the peptide to inhibit the loss in the kidney, has been used.
PEG non-specifically binds to a specific site or various sites of a target peptide to give an effect of increasing the molecular weight of a peptide, and thus inhibiting the loss by the kidney, and preventing hydrolysis, without causing any side-effects. For example, International Pat. Publication No. WO 2006/076471 describes that PEG binds to a B-type natriuretic peptide, or BNP, which binds to NPR-A to activate the production of cGMP, which leads to reduction in the arterial blood pressure, and as a result, is used as congestive heart failure therapeutic agent, thereby sustaining the physiological activity. U.S. Pat. No. 6,924,264 describes that PEG binds to the lysine residue of an exendin-4 to increase its in-vivo residence time. However, this method increases the molecular weight of PEG, thereby increasing the in-vivo residence time of the peptide drug, while as the molecular weight is increased, the titer of the peptide drug is remarkably reduced, and the reactivity with the peptide is also reduced. Accordingly, it undesirably lowers the yield.
International Pat. Publication No. WO 02/46227 describes a fusion protein prepared by coupling GLP-1, an exendin-4, or an analog thereof with human serum albumin or an immunoglobulin region (Fc) using a genetic recombination technology. U.S. Pat. No. 6,756,480 describes an Fc fusion protein prepared by coupling a parathyroid hormone (PTH) and an analog thereof with Fc region. These methods can address the problems such as low pegylation yield and non-specificity, but they still have a problem in that the effect of increasing the blood half-life is not noticeable as expected, and sometimes the titers are also low. In order to maximize the effect of increasing the blood half-life, various kinds of peptide linkers are used, but an immune response may be possibly caused. Further, if a peptide having disulfide bonds, such as BNP is used, there is a high probability of misfolding. As a result, such peptide can hardly be used.
In addition, a GLP-1 derivative, NN2211, is prepared by substitution of the amino acid of GLP-1, and is bound to an acyl side chain to form a non-covalent bond with albumin, thereby increasing its in-vivo residence time. However, it has a half-life of 11 to 15 hours, which does not indicate remarkable increase in the half-lives, as compared with the exendin-4. Thus, the GLP-1 derivative still needs to be injected once a day (Nauck et al., 2004). Further, CJC-1131 is a GLP-1 derivative having a maleimide reactive group for covalently binding the GLP-1 with albumin in blood, and efforts had been tried to develop the CJC-1131 for the purpose of increasing the in-vivo half-life, but such efforts were now stopped. A subsequently suggested substance, CJC-1134, is an exendin-4 which covalently binds to a recombinant albumin, and did not exhibit a remarkable effect of increasing blood stability, with the blood half-life being about 17 hours (Rat) (Thibauoleau et. al., 2006).